Summary: The goal of this project is to apply and develop molecular biological methods that can ensure that vaccines and cell substrates are free from viral, and particularly retroviral, contamination. Reverse transcriptase (RT) assays measure the presence of retroviruses. The conventional RT assay was insenstive and not quantitative. This changed with the advent of PCR-based RT PBRT) assays, which are a million-fold more sensitive than conventional RT assays. We set up the three PBRT assays at CBER in order to compare their sensitivities, specificities, and reproducibilities. Modifications were made to the assays to eliminate one problem with the assays, viz., their high background signals. We also modified the assay such that RT activities of cellular DNA polymerases were substrantially reduced. Recently, we have adapted the PBRT assay for use with the real time quantitative system, the TaqMan system, for use with the Perkin-Elmer 7700 system. This modified assay, the TM-PERT assay, is linear over at least 6 orders magnitude and is as sensitive as the original assays. With this sensitive TM-PERT assay, we have undertaken collaborative studies with two groups at CBER to apply the assay to several regulatory issues. In a study with Kurt Brorson and colleagues (OTRR/DMA/LMDI), we are assessing whether the TM-PERT assay can be used to monitor viral clearance during the preparation of monoclonal antibodies. In a separate study with Carolyn Wilson, we determined the RT level in porcine factor VIII, a therapeutic used in those hemophiliacs when human factor VIII is unsuitable. Because the product is pig derived and because all pig cells have and may express an endogenous retrovirus (PERV), the potential exists that such factor VIII preparations have PERV. All factor VIII lots tested had low levels of PERV as assessed by the TM-PERT assay by us and by RT-PCR by Dr Wilson. Dr Wilson showed that these preparations do not contain infectious virus. As part of our investigation into the chick RT activity, we began a study to determine whether pseudotypes can form between a retrovirus core and an envelope glycoprotein (Env) from paramyxoviruses or orthomyxoviruses. If so, then this may provide a means by which the chick RT particle could enter human cells. As a model system, we investigated measles, mumps and influenza virus Envs with HIV core particles. Our results show that pseudotypes can form in vitro, and thus there is a theoretical possibility of such pseudotypes forming in vivo. The consequences of such pseudotype formation remains unknown. We have demonstrated that mixed pseudotypes [i.e., pseudotypes containing envelope glycoproteins (Envs) from two different viruses] can form between measles virus and HIV-1 Envs. This raises the possibility that such virions can form in vivo. This may have implications for HIV pathogenesis, since HIV particles containing an Env of measles virus would be able to infect non-CD4 cells; such infected cells would have unknown consequences on HIV pathogenesis. We have initiated studies to develop quantitative assays to assess the biological activity of residual cell-substrate DNA. For many years, DNA resulting from cell substrates has been considered to pose a risk to vaccine recipients receiving products manufactured in neoplastic cells. This is one of the main reasons for these cells not being used for vaccine production to date. The risk is perceived to be either from an oncogenic potential and an infectious potential. We are developing quantitaive assays to assess both types of risks. We are developing Q-PCR assays to detect the genomes of primate polyomaviruses (SV40, JCV, BKV). Developing assays for these viruses will be a first step in our program to establish quantitative assays for the detection of adventitious agents.